Biological assays allow the detection of target molecules in a biological sample. Typically, the detection of target molecules is performed by using solid surfaces (e.g. microarrays or bottom of wells) or nanocarrier or microcarrier structures that are functionalized with detection molecules (ligands) designed to bind to specific targets.
One challenge of the biological assay technologies is the acceleration of mass transfer taking place during an assay. The problem of mass transfer is further exacerbated in multiplexed assays, where multiple target molecules are sought simultaneously in a single biological sample since the relative density of each probe is lower than in a single assay.
In order to overcome the limitations of mass transfer, different setups were described such as performing the multiplexed assays in a microchannel, thereby reducing the diffusion distance between the targets and the probes. For example, J. K.-K. Ng et al (2007), Anal. Chem Acta 582, pp. 295-303, describe a microfluidic device comprising microbeads being functionalized with oligonucleotides via biotin-streptavidin binding. The microfiuidic device consists of a broad chamber with a varying section and with a weir to trap the microbeads in a monolayered arrangement. Different sets of microbeads are sequentially introduced separated by unfunctionalized spacer beads. As can be seen from FIG. 5a in the document, the microbeads form large groups with undefined boundaries due to particle mixing with particles of the spacer sets. Since the beads have no characteristic which distinguishes them from each other, such as size, shape or a code, the boundaries of the different sets are unknown and only become revealed after the assay by the detection of the presence of the analyte in the sample. Therefore, the setup described by J. K.-K. Ng et al is not suited for multiplexed assays, as it is not possible to reliably determine the presence or absence of several targets in a sample. For example, in the absence of several analytes in the sample which correspond to consecutive sets, no signal will be recorded in an entire portion of the microchannel. It will thus be difficult to establish how many sets this portion actually corresponds to (thus there will be no indication on how many analytes are actually absent). It will also be difficult or even impossible to establish the identity of the subsequent analytes that react with the consecutive sets since the position in the sequence of these sets cannot be established reliably.
EP1712282A2, WO00/061198A1 and WO04/025560A1 describe setups having microcarrier elements placed inside microchannels such as their movement is restricted in the microchannel. Assays are performed by flowing fluids through. This type of setup is effective for mass transfer since diffusion distances are small and the movement of the sample relative to the microcarriers brings the target molecules to the proximity of the receptor molecules. This type of setups also reduces cost by reducing the amount of reagents that are needed.
However, in EP1712282A2 and WO00/061198A1, the order of the microcarriers in the microchannel is very important since it defines the identity of the microcarriers. In WO2004025560A1, the microcarriers are encoded so that their order in the microchannel is not as critical as in EP1712282A2. Still, the disclosure of WO2004025560A1 only describes configurations where the microcarriers are strictly aligned behind each other in order to meet the requirements of the proposed decoding mechanism that requires a specific placement of the microcarriers' codes for allowing their identification.
EP1712282A2, WO00/061198A1 and WO04/025560A1 describe setups that are not easy to prepare in practice because they require a very controlled introduction of microcarriers in a confined space, either to control their order or to align them for the decoding purpose. To achieve such configurations, specialized methods and specific settings involving microscopy, micromanipulation (use of microscopically controlled forces) and/or microfabrication techniques are required.
Indeed, the microcarriers need to be introduced in the microchannel by some process that involves either intricate micromanipulation of individual microcarriers such as described in WO0061198A1 or, when the exact position of each microcarrier does not need to be controlled, some kind of funnel mechanism that guides them from a bulk into a small microchannel such as described in WO04/025560A1. The funnel mechanism is simpler to build in practice but is sensitive to clogging by forming arches in the entry of the microchannel 1 (FIGS. 13 and 14). Further to the funnel approach, WO04/025560A1 suggests the production of assay sticks by a sandwich approach wherein the beads are placed on a lower plate having grooves. Subsequently, an upper plate is laid on top and attached to the lower plate.
One practical consequence of the level of sophistication required to prepare the setups described in the prior art is that it reduces the possibility of being used to produce flexible configuration for research use by a laboratory technician. For example, it would be very difficult to allow the preparation of custom-made configurations by a laboratory technician that would like to use its own biochemical coating procedures on microcarriers (for example to test biological probes that are in development) and then introduce them in the setup to perform biological assays.
Therefore, there is a need in the art for assay devices and methods which improve the mass transfer in biological multiplexing assays based on microcarriers and simplify the overall procedure for preparing the setup, executing the biological assay and performing the necessary readouts.